The human bacterial pathogen group A Streptococcus (GAS) causes a broad spectrum of diseases, including pharyngitis, necrotizing fasciitis, and a toxic-shock-like syndrome. The long-term aim of the proposed research is to advance our understanding of the global regulatory mechanisms controlling bacterial virulence. Newly identified virulence-related regulatory mechanisms would represent attractive targets for manipulation by novel antimicrobial agents, and thus enhance public health. Small regulatory RNAs (sRNAs) represent a poorly understood area of gene regulation in GAS and related pathogens. The specific hypothesis is that sRNAs represent a major constituent of the global regulatory networks coordinating GAS virulence. This hypothesis is based upon the following observations. First, sRNAs regulate virulence in some bacterial pathogens. Second, a bioinformatic analysis indicates that GAS encodes 56 putative sRNAs. Third, two sRNAs have been experimentally verified in GAS and both, through as-yet-unknown mechanisms, appear to regulate virulence factor expression. Our hypothesis will be tested by the following specific aims: 1. Test the hypothesis that sRNAs are prevalent in GAS and conserved across streptococcal species. Whether sRNAs represent a major mechanism of regulation in GAS will be investigated through use of a custom Affymetrix microarray to detect sRNAs transcribed under a series of experimental conditions mimicking human infections, including both ex vivo and in vivo disease models. Completion of this specific aim would represent the first genome-wide analysis of sRNAs within a pathogen from the order Lactobacillales, which includes the streptococci and enterococci, and stimulate sRNA research in a spectrum of human bacterial pathogens. 2. Test the hypothesis that sRNAs regulate GAS virulence. Candidate sRNAs will be triaged based upon sequence conservation across Lactobacillales pathogens, and also upon sRNA expression patterns during in vivo growth. Isogenic sRNA mutant strains will be constructed for 12 conserved sRNAs and assayed in vitro and ex vivo for altered virulence characteristics. Strains with altered virulence characteristics will subsequently be tested for attenuated virulence in two mouse models of invasive disease. Completion of this specific aim would identify sRNAs critical for normal progression of GAS infections, and provide impetus to delineate the pathways by which these regulators function, with the ultimate goal of inhibiting these pathways through use of novel antimicrobials. PUBLIC HEALTH RELEVANCE: Each year in the U.S. there are ~30 million cases of GAS pharyngitis. The proposed research would provide insight into a fundamental and yet understudied field of gene regulation in GAS and related pathogens, and provide the research community with a detailed blueprint of sRNA locations and expression patterns to stimulate further study. Public health may be enhanced through the long-term goal of translating knowledge of sRNA virulence-regulating pathways into new treatment and/or preventative regimes based upon the inhibition of these pathways by novel antimicrobial agents.